We propose to advance a technology that, for the first time, will permit in vivo detection of any neurotransmitter that binds to a G-protein coupled receptor. This includes the monoamines, which play a fundamental role in neuromodulation and a biomedical role in addiction and mental disorders, and peptide transmitters, which are important for the neuroendocrine system and control of vascular tone and blood flow. At present, only limited in vivo assays are available to detect monoamines and peptide neurotransmission. This deficit is a major impediment to understanding normal signaling in the brain. To fill this gap of missing, we introduce Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for the optical measurement of exogenous receptor activity in vivo. A CNiFER is a clonal cell-line that is engineered to express a specific metabotropic receptor that couples to G proteins and a genetically encoded FRET-based Ca2+ sensor that detects changes in intracellular [Ca2+]. Stimulation of the metabotropic receptor leads to elevations in cytosolic [Ca2+], providing a direct and rapid readout of local neurotransmitter activity. CNiFERs are acutely or chronically implanted and fluorescence measured with in vivo two-photon microscopy. This new technology will make it possible to map the spatial patterns of in vivo signaling with up to ~ 100 5M spatial precision and ~ 1 s temporal resolution. PUBLIC HEALTH RELEVANCE: Kleinfeld, David and Slesinger, Paul A. Chemical communication between brain cells underlies the computations performed by a nervous system as animals' locomote and interact with their environment. Yet measurements of the spatial patterns and temporal release of the neuronal transmitters and modulators that mediate this communication have proven to be difficult. Here we propose to develop and implement a new technology, CNiFERs (cell-based neurotransmitter fluorescent engineered reporters), that may be used in conjunction with optical microscopy as a means to detect patterns of neuronal and neurovascular signaling patterns in the brain in vivo.